Adenosine triphosphate (ATP) conversion to ADP is a central process in all living organisms and is catalyzed by a vast number of different enzymes. The energy generated by ATP hydrolysis can drive metabolic processes, directed transport, force-generation and movement as well as signal transduction and regulation. Kinases transfer the terminal phosphate of ATP yielding ADP as a product and phosphorylate a wide variety of substrates, from metabolic intermediates to proteins, so controlling their activity. Hence, assays to monitor ADP concentrations have wide applications in biochemical and biomedical research, ranging from detailed understanding of mechanochemical coupling in motor proteins to screening for ATPase and kinase inhibitors.
Despite the importance of ADP detection in biological systems only few methods for monitoring ADP concentrations are available to date. Most widely applied is a coupled enzyme assay using pyruvate kinase and lactate dehydrogenase, which couples ADP generation to the oxidation of NADH and a concomitant absorbance or fluorescence decrease. Although frequently used to study steady-state kinetics of ATPases and kinases, this approach is generally not suitable for mechanistic studies based on transient kinetics, since it lacks fast response and high sensitivity, which are drawbacks of this system. The fact that several components must be present for the assay is also problematic, and adds complexity and cost, especially for high throughput applications. Some compounds may interfere with one of the assay components and/or with UV detection. With a large number of components, the risks of such interference problems are correspondingly large.
Recently, the assay has been modified to generate a fluorescence by coupling the pyruvate kinase reaction with pyruvate oxidase and horseradish peroxidase (1). Amplex Red is converted to Resorufin by peroxidase, yielding a fluorescence increase at 590 nm. The fluorescence detection at high wavelength provides improvements, both in enhanced sensitivity as well as in separating the optical signal from the absorbance of many compounds. However, this assay does not circumvent the problem of interference with one of the several assay components. Moreover, the assay is still an enzyme-coupled assay which imposes requirements on the system to be permissive of the differing enzyme activities required, and involves numerous interdependent components to the assay, which remain problems even with this improved version.
Aimed at the development of high throughput assays for kinases, two ADP-specific sensors have been reported, which are based on ADP recognition by RNA molecules (2). The first relies on an RNA aptamer which selectively binds ADP and can be used to monitor ADP generation in a (radiometric) scintillation proximity assay. Although this method can be used for real time measurements, it is again a complex multi-component system and involves radiometric scintillation, which is hazardous and costly. The second sensor creates a fluorescence readout based on ADP dependent self-cleavage of a fluorescently labeled ribozyme. This method cannot be used for real time measurements, which is a hindrance.
An alternative approach is to take advantage of the highly specific interaction of a binding protein with the target molecule. By attaching a fluorophore in a suitable position on the protein, ligand recognition can be coupled to an optical signal. Such fluorescent protein-based biosensors have been reported for a number of biomolecules such as sugars, amino acids, metal ions and phosphate (3-7). One advantage of this type of sensor is that the signal change can be very fast, only limited by the speed of ligand binding or the associated conformational change. Furthermore, only a single component, the labeled protein, is needed for detection, so they are also classified as reagentless biosensors. Such a biosensor for ADP has been developed previously, based on a nucleoside diphosphate kinase labeled with the coumarin dye IDCC (8). The biosensor responds to the ADP/ATP concentration ratio and thus can detect a wide range of ADP concentrations, from sub-micromolar to millimolar. However, the fluorescence decreases with ADP and this is unfavourable. Moreover, decreasing fluorescence with ADP binding causes the sensitivity to be low, particularly when measuring initial rates, which is a problem. In addition, the fluorescence intensity is not linearly dependent on the ADP concentration, which is a drawback of this system.
The present invention seeks to overcome problem(s) associated with the prior art.